Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Cryo-EM structures of human RAD51 recombinase filaments during catalysis of DNA-strand exchange

Nature Structural & Molecular Biology volume 24, pages 40–46 (2017)Cite this article

Subjects

Abstract

The central step in eukaryotic homologous recombination (HR) is ATP-dependent DNA-strand exchange mediated by the Rad51 recombinase. In this process, Rad51 assembles on single-stranded DNA (ssDNA) and generates a helical filament that is able to search for and invade homologous double-stranded DNA (dsDNA), thus leading to strand separation and formation of new base pairs between the initiating ssDNA and the complementary strand within the duplex. Here, we used cryo-EM to solve the structures of human RAD51 in complex with DNA molecules, in presynaptic and postsynaptic states, at near-atomic resolution. Our structures reveal both conserved and distinct structural features of the human RAD51–DNA complexes compared with their prokaryotic counterpart. Notably, we also captured the structure of an arrested synaptic complex. Our results provide new insight into the molecular mechanisms of the DNA homology search and strand-exchange processes.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structures of RAD51 presynaptic and postsynaptic complexes.
Figure 2: Structure analysis of the RAD51 protomer-protomer interface.
Figure 3: Interaction of RAD51 with DNA in the presynaptic and postsynaptic complexes.
Figure 4: Capture of the arrested state in Rad51-mediated DNA-strand exchange.
Figure 5: Model of RAD51-mediated DNA-strand exchange, with the intermediate state depicted.

Similar content being viewed by others

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Referenced accessions

Electron Microscopy Data Bank

Protein Data Bank

References

  1. Wang, A.T. et al. A dominant mutation in human RAD51 reveals its function in DNA interstrand crosslink repair independent of homologous recombination. Mol. Cell 59, 478–490 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Mazón, G., Mimitou, E.P. & Symington, L.S. SnapShot: homologous recombination in DNA double-strand break repair. Cell 142, 648.e1–648.e2 (2010).

    Article  CAS  Google Scholar 

  3. Lambert, S. et al. Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange. Mol. Cell 39, 346–359 (2010).

    Article  CAS  PubMed  Google Scholar 

  4. van den Bosch, M., Lohman, P.H.M. & Pastink, A. DNA double-strand break repair by homologous recombination. Biol. Chem. 383, 873–892 (2002).

    CAS  PubMed  Google Scholar 

  5. Takata, M. et al. Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J. 17, 5497–5508 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Da Ines, O., Gallego, M.E. & White, C.I. Recombination-independent mechanisms and pairing of homologous chromosomes during meiosis in plants. Mol. Plant 7, 492–501 (2014).

    Article  CAS  PubMed  Google Scholar 

  7. Habu, T., Taki, T., West, A., Nishimune, Y. & Morita, T. The mouse and human homologs of DMC1, the yeast meiosis-specific homologous recombination gene, have a common unique form of exon-skipped transcript in meiosis. Nucleic Acids Res. 24, 470–477 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Krejci, L., Altmannova, V., Spirek, M. & Zhao, X. Homologous recombination and its regulation. Nucleic Acids Res. 40, 5795–5818 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Sung, P. & Klein, H. Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat. Rev. Mol. Cell Biol. 7, 739–750 (2006).

    Article  CAS  PubMed  Google Scholar 

  10. Wright, W.D. & Heyer, W.D. Rad54 functions as a heteroduplex DNA pump modulated by its DNA substrates and Rad51 during D loop formation. Mol. Cell 53, 420–432 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Galkin, V.E. et al. The Rad51/RadA N-terminal domain activates nucleoprotein filament ATPase activity. Structure 14, 983–992 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Gupta, R.C., Bazemore, L.R., Golub, E.I. & Radding, C.M. Activities of human recombination protein Rad51. Proc. Natl. Acad. Sci. USA 94, 463–468 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Benson, F.E., Stasiak, A. & West, S.C. Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA. EMBO J. 13, 5764–5771 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Seitz, E.M., Brockman, J.P., Sandler, S.J., Clark, A.J. & Kowalczykowski, S.C. RadA protein is an archaeal RecA protein homolog that catalyzes DNA strand exchange. Genes Dev. 12, 1248–1253 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Yang, S., Yu, X., Seitz, E.M., Kowalczykowski, S.C. & Egelman, E.H. Archaeal RadA protein binds DNA as both helical filaments and octameric rings. J. Mol. Biol. 314, 1077–1085 (2001).

    Article  CAS  PubMed  Google Scholar 

  16. Hilario, J., Amitani, I., Baskin, R.J. & Kowalczykowski, S.C. Direct imaging of human Rad51 nucleoprotein dynamics on individual DNA molecules. Proc. Natl. Acad. Sci. USA 106, 361–368 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Galletto, R., Amitani, I., Baskin, R.J. & Kowalczykowski, S.C. Direct observation of individual RecA filaments assembling on single DNA molecules. Nature 443, 875–878 (2006).

    Article  CAS  PubMed  Google Scholar 

  18. Register, J.C. III,, Christiansen, G. & Griffith, J. Electron microscopic visualization of the RecA protein-mediated pairing and branch migration phases of DNA strand exchange. J. Biol. Chem. 262, 12812–12820 (1987).

    Article  CAS  PubMed  Google Scholar 

  19. Ragunathan, K., Joo, C. & Ha, T. Real-time observation of strand exchange reaction with high spatiotemporal resolution. Structure 19, 1064–1073 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Mani, A., Braslavsky, I., Arbel-Goren, R. & Stavans, J. Caught in the act: the lifetime of synaptic intermediates during the search for homology on DNA. Nucleic Acids Res. 38, 2036–2043 (2010).

    Article  CAS  PubMed  Google Scholar 

  21. Folta-Stogniew, E., O'Malley, S., Gupta, R., Anderson, K.S. & Radding, C.M. Exchange of DNA base pairs that coincides with recognition of homology promoted by E. coli RecA protein. Mol. Cell 15, 965–975 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Danilowicz, C. et al. The differential extension in dsDNA bound to Rad51 filaments may play important roles in homology recognition and strand exchange. Nucleic Acids Res. 42, 526–533 (2014).

    Article  CAS  PubMed  Google Scholar 

  23. Danilowicz, C. et al. RecA homology search is promoted by mechanical stress along the scanned duplex DNA. Nucleic Acids Res. 40, 1717–1727 (2012).

    Article  CAS  PubMed  Google Scholar 

  24. Li, X. et al. Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics. Nucleic Acids Res. 35, 4124–4140 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Sanchez, H., Kertokalio, A., van Rossum-Fikkert, S., Kanaar, R. & Wyman, C. Combined optical and topographic imaging reveals different arrangements of human RAD54 with presynaptic and postsynaptic RAD51-DNA filaments. Proc. Natl. Acad. Sci. USA 110, 11385–11390 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. van der Heijden, T. et al. Homologous recombination in real time: DNA strand exchange by RecA. Mol. Cell 30, 530–538 (2008).

    Article  CAS  PubMed  Google Scholar 

  27. Wu, Y., He, Y., Moya, I.A., Qian, X. & Luo, Y. Crystal structure of archaeal recombinase RADA: a snapshot of its extended conformation. Mol. Cell 15, 423–435 (2004).

    Article  CAS  PubMed  Google Scholar 

  28. Pellegrini, L. et al. Insights into DNA recombination from the structure of a RAD51–BRCA2 complex. Nature 420, 287–293 (2002).

    Article  CAS  PubMed  Google Scholar 

  29. Conway, A.B. et al. Crystal structure of a Rad51 filament. Nat. Struct. Mol. Biol. 11, 791–796 (2004).

    Article  CAS  PubMed  Google Scholar 

  30. Story, R.M., Weber, I.T. & Steitz, T.A. The structure of the E. coli recA protein monomer and polymer. Nature 355, 318–325 (1992).

    Article  CAS  PubMed  Google Scholar 

  31. Yu, X., Jacobs, S.A., West, S.C., Ogawa, T. & Egelman, E.H. Domain structure and dynamics in the helical filaments formed by RecA and Rad51 on DNA. Proc. Natl. Acad. Sci. USA 98, 8419–8424 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. VanLoock, M.S. et al. ATP-mediated conformational changes in the RecA filament. Structure 11, 187–196 (2003).

    Article  CAS  PubMed  Google Scholar 

  33. Egelman, E.H. Does a stretched DNA structure dictate the helical geometry of RecA-like filaments? J. Mol. Biol. 309, 539–542 (2001).

    Article  CAS  PubMed  Google Scholar 

  34. Chen, Z., Yang, H. & Pavletich, N.P. Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structures. Nature 453, 489–494 (2008).

    Article  CAS  PubMed  Google Scholar 

  35. Zhao, W. & Sung, P. Significance of ligand interactions involving Hop2-Mnd1 and the RAD51 and DMC1 recombinases in homologous DNA repair and XX ovarian dysgenesis. Nucleic Acids Res. 43, 4055–4066 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Cho, N.W., Dilley, R.L., Lampson, M.A. & Greenberg, R.A. Interchromosomal homology searches drive directional ALT telomere movement and synapsis. Cell 159, 108–121 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Egelman, E.H. The iterative helical real space reconstruction method: surmounting the problems posed by real polymers. J. Struct. Biol. 157, 83–94 (2007).

    Article  CAS  PubMed  Google Scholar 

  38. Short, J.M. et al. High-resolution structure of the presynaptic RAD51 filament on single-stranded DNA by electron cryo-microscopy. Nucleic Acids Res. 44, 9017–9030 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Forget, A.L., Loftus, M.S., McGrew, D.A., Bennett, B.T. & Knight, K.L. The human Rad51 K133A mutant is functional for DNA double-strand break repair in human cells. Biochemistry 46, 3566–3575 (2007).

    Article  CAS  PubMed  Google Scholar 

  40. Flott, S. et al. Regulation of Rad51 function by phosphorylation. EMBO Rep. 12, 833–839 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Amunugama, R. et al. RAD51 protein ATP cap regulates nucleoprotein filament stability. J. Biol. Chem. 287, 8724–8736 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Reymer, A., Frykholm, K., Morimatsu, K., Takahashi, M. & Nordén, B. Structure of human Rad51 protein filament from molecular modeling and site-specific linear dichroism spectroscopy. Proc. Natl. Acad. Sci. USA 106, 13248–13253 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Prasad, T.K., Yeykal, C.C. & Greene, E.C. Visualizing the assembly of human Rad51 filaments on double-stranded DNA. J. Mol. Biol. 363, 713–728 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. Shinohara, T. et al. Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair. Nucleic Acids Res. 43, 973–986 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Qi, Z. et al. DNA sequence alignment by microhomology sampling during homologous recombination. Cell 160, 856–869 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Cloud, V., Chan, Y.L., Grubb, J., Budke, B. & Bishop, D.K. Rad51 is an accessory factor for Dmc1-mediated joint molecule formation during meiosis. Science 337, 1222–1225 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Chi, P., Van Komen, S., Sehorn, M.G., Sigurdsson, S. & Sung, P. Roles of ATP binding and ATP hydrolysis in human Rad51 recombinase function. DNA Repair (Amst.) 5, 381–391 (2006).

    Article  CAS  Google Scholar 

  48. Sigurdsson, S., Trujillo, K., Song, B., Stratton, S. & Sung, P. Basis for avid homologous DNA strand exchange by human Rad51 and RPA. J. Biol. Chem. 276, 8798–8806 (2001).

    Article  CAS  PubMed  Google Scholar 

  49. Chi, P., San Filippo, J., Sehorn, M.G., Petukhova, G.V. & Sung, P. Bipartite stimulatory action of the Hop2-Mnd1 complex on the Rad51 recombinase. Genes Dev. 21, 1747–1757 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Abrishami, V. et al. Alignment of direct detection device micrographs using a robust Optical Flow approach. J. Struct. Biol. 189, 163–176 (2015).

    Article  PubMed  Google Scholar 

  51. Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Mindell, J.A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003).

    Article  PubMed  Google Scholar 

  53. Scheres, S.H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Clemens, D.L., Ge, P., Lee, B.Y., Horwitz, M.A. & Zhou, Z.H. Atomic structure of T6SS reveals interlaced array essential to function. Cell 160, 940–951 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199 (1996).

    Article  CAS  PubMed  Google Scholar 

  56. van Heel, M., Harauz, G., Orlova, E.V., Schmidt, R. & Schatz, M. A new generation of the IMAGIC image processing system. J. Struct. Biol. 116, 17–24 (1996).

    Article  CAS  PubMed  Google Scholar 

  57. Ge, P. & Zhou, Z.H. Hydrogen-bonding networks and RNA bases revealed by cryo electron microscopy suggest a triggering mechanism for calcium switches. Proc. Natl. Acad. Sci. USA 108, 9637–9642 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Grigorieff, N. FREALIGN: high-resolution refinement of single particle structures. J. Struct. Biol. 157, 117–125 (2007).

    Article  CAS  PubMed  Google Scholar 

  59. Zhang, R. & Nogales, E. A new protocol to accurately determine microtubule lattice seam location. J. Struct. Biol. 192, 245–254 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Stein, N. CHAINSAW: a program for mutating pdb files used as templates in molecular replacement. J. Appl. Crystallogr. 41, 641–643 (2008).

    Article  CAS  Google Scholar 

  61. Pettersen, E.F. et al. UCSF Chimera: a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  62. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  CAS  PubMed  Google Scholar 

  63. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Yan, C. et al. Structure of a yeast spliceosome at 3.6-angstrom resolution. Science 349, 1182–1191 (2015).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank J.L. Lei, Y.J. Xu and T. Yang for their support in cryo-EM; the high-performance computation facility at the National Protein Science Facility (Beijing) at Tsinghua University; X. Li for help in data collection; E. Egelman for advice in setting up the IHRSR algorithm; T. Baker for distributing the IHRSR scripts in the SPIDER package; and P. Ge and Z.H. Zhou for distributing the IHRSR-incorporated version of RELION1.2. We give special thanks to C. Yan for help in building the atomic models. This work was supported by funding from the National Science Foundation of China (grant 31270765), the Key Research and Development Program of MOST (grant 2016YFA0501100) and the Beijing Municipal Science & Technology Commission (grant Z161100000116034) to H.-W.W., and funding from the US National Institutes of Health (grants CA168635, ES007061 and ES015252) to P.S.

Author information

Author notes

  1. Jingfei Xu, Lingyun Zhao, Yuanyuan Xu and Weixing Zhao: These authors contributed equally to this work.

Authors and Affiliations

  1. Ministry of Education Key Laboratory of Protein Sciences, Tsinghua-Peking Joint Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Jingfei Xu, Lingyun Zhao & Hong-Wei Wang

  2. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA

    Yuanyuan Xu, Weixing Zhao & Patrick Sung

Authors
  1. Jingfei Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lingyun Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yuanyuan Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Weixing Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Patrick Sung
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hong-Wei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.-W.W. conceived the project. H.-W.W. and P.S. designed the experiments. J.X., L.Z. and Y.X. performed EM and structural determination. W.Z. generated key research materials and performed biochemical assays. J.X., L.Z., P.S. and H.-W.W. wrote the manuscript.

Corresponding authors

Correspondence to Patrick Sung or Hong-Wei Wang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Cryo-EM analysis of assembly of the RAD51 presynaptic and postsynaptic complexes.

(a) Purified human RAD51 and mouse Hop2-Mnd1 were analyzed by SDS-PAGE with Coomassie blue staining and by DNA strand exchange assays. The reaction scheme setup is illustrated. “+” stands for the concentration of Hop2-Mnd1 and +, ++, +++ means 100 nM, 200 nM, and 400 nM, respectively. Rad51 concentration was kept constant at 2 μM in all the assays. The mean values ± s.d. from three independent experiments were presented as bar diagram on the right panel. (b) A representative cryo-EM micrograph of presynaptic filaments and a representative 2D class average are shown in the left panel. 3D reconstruction of the presynaptic filament via IHRSR is shown on the right. (c) A representative cryo-EM micrograph of postsynaptic filaments and a representative 2D class average are shown in the left panel. 3D reconstruction of the postsynaptic complex is shown on the right. (d) 3D reconstruction of the RAD51-ssDNA presynaptic complex incubated with Hop2-Mnd1 is shown on the left panel. The difference map between 3D reconstructions of RAD51-ssDNA presynaptic complex with and without Hop2-Mnd1 incubation is shown in the middle and the right panels with inverted contrast separately. The corresponding atomic model of the postsynaptic complex is shown as reference.

Supplementary Figure 2 Structure determination of presynaptic and postsynaptic complexes.

(a) Work flow for 3D reconstruction of the presynaptic and postsynaptic complexes using two alternative strategies. (b) and (d) show respectively the Fourier shell correlation (FSC) curves of the presynaptic and postsynaptic complexes between maps calculated from two independent half datasets. (c) and (e) show respectively the FSC curves of the presynaptic and postsynaptic complexes between the built atomic models and the 3D density maps (black, final refined atomic model versus final map; blue, final refined model versus working map; red, final model versus testing map).

Supplementary Figure 3 Representative EM densities with the corresponding atomic models.

(a) The ssDNA and dsDNA selected from presynaptic complex and postsynaptic complex, respectively. (b) Selected three α-helix elements of RAD51. (c) Selected 4 β-strand elements of RAD51. (d) The β-sheet motif in the middle of RAD51’s ATPase core.

Supplementary Figure 4 Structural comparison of RAD51 and orthologs in their nucleotide-binding sites at the interface between protomers.

(a) RAD51 protomers from the presynaptic and postsynaptic complexes are superimposed, colored by the RMSD value between the two atomic models. (b) The presynaptic filaments of human RAD51 (green and light green for two protomers) and yeast Rad51 filament (salmon) are superimposed with the models of lower protomers aligned to each other. (c) The superimposition of presynaptic filaments of human RAD51 (green and light green for two protomers) and E.coli RecA (grey) with the models of lower protomers aligned to each other. An enlarged view of the nucleotide binding pocket is shown on the right. (d) The superimposition of human RAD51 and Methanococus Voltae RadA (pink) with the models of the lower protomers aligned to each other. An enlarged view of the nucleotide binding pocket is shown on the right.

Supplementary Figure 5 Sequence alignment of human RAD51 and its orthologs.

The secondary structural elements of human RAD51 determined in our structure are labeled on top of the corresponding residues. Walker A and B motifs involved in nucleotide binding, the conserved Loop 1 and Loop 2 regions responsible for binding ssDNA or dsDNA, and key residues for human RAD51 function are all labeled. Y54 and F195 that are located at the interface between human RAD51 protomers are shown in green triangles. K133, T134, E163, and D316 that interact with AMP-PNP-Mg2+ are shown in red triangles. The blue triangles point to R229, R241, A271, V273, G288, G289, and N290 residues that are involved in ssDNA binding and also shown in Figure 2. The magenta triangles point to R235 and D274 that are involved in dsDNA binding and also shown in Figure 2.

Supplementary Figure 6 Cryo-EM and reconstruction algorithm of the arrested synaptic complex with hexameric repeating symmetry.

(a) A representative cryo-EM micrograph of the arrested synaptic complex. (b) Semitransparent rendering of the 3D reconstruction of the arrested synaptic complex via IHRSR using the helical parameters of RAD51 monomer as asymmetric unit. The postsynaptic complex atomic model is docked in the density for comparison. (c) Workflow of the reconstruction algorithm of the arrested synaptic complex with a hexameric repeating RAD51 as asymmetric unit. Particles were segmented from filaments with non-overlap of about 92 Å, corresponding to a hexameric rise. During refinement the spatial relationship between neighboring particles from the same filament was taken into account, as was the relationship for different filaments. Details of the algorithm are described in Online Methods.

Supplementary Figure 7 Structure analysis of the arrested synaptic complex.

(a) 3D reconstruction of the arrested synaptic complex via the algorithm described in Figure S6c. (b) and (c) show difference maps between the 3D reconstruction and the atomic models of the postsynaptic complex and presynaptic complex, respectively. (d) 3D reconstruction of the postsynaptic complex dataset using the same algorithm as for the arrested synaptic complex. (e) and (f) are difference maps between the 3D reconstruction and the atomic models of the postsynaptic and presynaptic complexes, respectively. Please be noted that the extra densities on the outer surface of RAD51’s N-terminal domains are due to the lack of the very N-terminal 21 residues in the atomic models. These extra densities are hidden in Figure 4b in order to show the extra densities near the nucleic acids more clearly. All the difference maps in (b), (c), (e), (f) are shown in a threshold of 8σ.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Table 1 (PDF 1551 kb)

The EM density map of the RAD51 presynaptic complex and its atomic model.

The cryo-EM density map of the RAD51 presynaptic complex is first shown in surface representation, followed by superimposition of 6-protomer-atomic model of the presynaptic complex. Two representative α-helices and the β-sheet motif in the ATPase core of RAD51 are shown in zoom-in views. The invading strand fitted within its corresponding density is shown by hiding RAD51 protomers. Subsequently, RAD51 residues that are involved in ssDNA interaction are illustrated in two views in the presynaptic complex. (MP4 25269 kb)

The EM density map of the RAD51 postsynaptic complex and its atomic model.

The cryo-EM density map in surface representation and superimposition of atomic model of the RAD51 post-synaptic complex are displayed and rotated. The complementary strand and invading strand corresponding to 6 protomers of RAD51 are shown fitted within the corresponding density. The AMP-PNP segmented from six RAD51 protomers are presented by hiding other elements. Subsequently, important residues, R235 and D274, for stabilizing the complementary strand are shown with their side chains fitting in the density map. (MP4 19614 kb)

The EM density map of the arrested state of the RAD51 synaptic complex and its difference maps with the atomic models.

The 3D reconstruction of the arrested synaptic complex is shown and rotated with docked atomic model of post-synaptic complex synchronously. Subsequently, the difference maps between the 3D reconstruction and the atomic models of the presynaptic complex and post-synaptic complex are shown separately and then in superimposition with each other as in Figure 4B. Please be noted that the extra densities on the outer surface of RAD51's N-terminal domains are due to the lack of the very N-terminal 21 residues in the atomic models. In the final superposition animation, these extra densities are hidden in order to show the extra densities near the nucleic acids more clearly. (MP4 16225 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Xu, J., Zhao, L., Xu, Y. et al. Cryo-EM structures of human RAD51 recombinase filaments during catalysis of DNA-strand exchange. Nat Struct Mol Biol 24, 40–46 (2017). https://doi.org/10.1038/nsmb.3336

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.3336

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing